Large-scale domain dynamics and adenosylcobalamin reorientation orchestrate radical catalysis in ornithine 4,5-aminomutase

Abstract

D-ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii converts D-ornithine to 2,4-diaminopentanoic acid by way of radical propagation from an adenosylcobalamin (AdoCbl) to a pyridoxal 5'-phosphate (PLP) cofactor. We have solved OAM crystal structures in different catalytic states that together demonstrate unusual stability of the AdoCbl Co-C bond and that radical catalysis is coupled to large-scale domain motion. The 2.0-A substrate-free enzyme crystal structure reveals the Rossmann domain, harboring the intact AdoCbl cofactor, is tilted toward the edge of the PLP binding triose-phosphate isomerase barrel domain. The PLP forms an internal aldimine link to the Rossmann domain through Lys(629), effectively locking the enzyme in this "open" pre-catalytic conformation. The distance between PLP and 5'-deoxyadenosyl group is 23 A, and large-scale domain movement is thus required prior to radical catalysis. The OAM crystals contain two Rossmann domains within the asymmetric unit that are unconstrained by the crystal lattice. Surprisingly, the binding of various ligands to OAM crystals (in an oxygen-free environment) leads to transimination in the absence of significant reorientation of the Rossmann domains. In contrast, when performed under aerobic conditions, this leads to extreme disorder in the latter domains correlated with the loss of the 5'-deoxyadenosyl group. Our data indicate turnover and hence formation of the "closed" conformation is occurring within OAM crystals, but that the equilibrium is poised toward the open conformation. We propose that substrate binding induces large-scale domain motion concomitant with a reconfiguration of the 5'-deoxyadenosyl group, triggering radical catalysis in OAM.

FIGURE 1.

A proposed mechanism for the reversible rearrangement of d-ornithine to 2,4-diaminopentanoic acid. In this mechanism, the PLP forms a Schiff base with d-ornithine prior to homolysis.

FIGURE 2.

Resting state OAM structure. A, the TIM barrel, Rossmann domain, and dimerization domain of two individual β subunits (OraE) within the α₂β₂ heterodimer are represented as blue and green ribbons, whereas the α subunits (OraS) are shown as yellow and cyan ribbons. AdoH, PLP, and cobalamin are shown as red, black, and magenta sticks, respectively. The red dashed lines represent disordered residues in the flexible loop connecting the Rossmann domain to the TIM barrel. B, the AdoCbl binding site for the Rossmann domain of monomer A in contact with the TIM barrel domain of monomer C is shown. The AdoCbl and selected residues are shown in atom colored sticks, with purple and cyan carbon atoms, respectively. The 2F_o − F_c electron density corresponding to the 5′-deoxyadenosine moiety is shown in a blue mesh (contoured at 1 σ). For comparison, the structure of the free AdoCbl has been superposed and is shown in black lines.

FIGURE 3.

Proposed conformational substates of OAM during catalysis. In the schematic the TIM barrel is represented by the blue box; Rossmann domain, green circle; AdoCbl, red parallelogram; PLP cofactor, yellow diamond; and the black line is the flexible hinge. Activated AdoCbl is colored pink. In the resting configuration, or “pre-catalytic” state, the PLP is tethered to the Rossmann domain by an external aldimine link and located ∼23 Å from deoxyadenosyl group of AdoCbl. Substrate binding is envisioned to release the internal aldimine link (orange line) to free the Rossmann domain to “sample” different conformations to find a suitable position for hydrogen atom abstraction from the PLP-bound substrate. Once in a suitable configuration, steps 2–7 of the catalytic cycle shown in Fig. 1 (homolysis, H-atom abstraction and radical-based isomerization and geminate recombination) occur to form the product bound PLP intermediate. Following release of product, the internal aldimine link is reformed with return to the resting form of the enzyme. The gray scale figure represents the form of the enzyme following reaction of cob(II)alamin intermediate with O₂ and formation of hydroxycobalamin. The asterisks denote the conformational substates of OAM for which we have x-ray crystal structures.

FIGURE 4.

Ornithine binding site. The covalent adduct made between ornithine and PLP can be readily observed in anaerobic, ornithine-soaked OAM crystals. Selected residues of the active site of chain A are shown in atom colored stick with cyan carbons. The PLP and ornithine moieties are shown with green and purple carbons, respectively. Key interactions made between ornithine and various residues are shown in black dotted lines. The 2F_o − F_c electron density corresponding to the covalent substrate adduct is shown as a blue mesh (contoured at 1 σ).

FIGURE 5.

Ligand soaks under aerobic conditions. A, electron density corresponding to the unconstrained Rossmann domains is missing following soaking with DAB under anaerobic conditions. The electron density corresponding to the OAM heterodimer made of chains A and C is shown as a blue mesh (contoured at 1 σ), with Rossmann domains depicted in red. B, electron density corresponding to the 5′-deoxyadenosine moiety is missing for unconstrained Rossmann domains following ligand soaks under aerobic conditions with removal of excess ligand (allowing the Lys⁶²⁹-PLP covalent bond to be reformed). Selected residues of the AdoCbl binding site and the AdoCbl cofactor are shown in atom colored sticks for Rossmann domains D and B, respectively.

FIGURE 6.

Proposed model of OAM in the open and closed conformation. The TIM barrel and Rossmann domain from the substrate-free OAM structure are gray and blue, respectively. The red schematic is a model (based on superposition with GM (PDB code 1I9C)) of the OAM Rossmann domain orientated over the TIM barrel, in a position that would facilitate hydrogen transfer between AdoCbl and the PLP-bound substrate. The substrate-bound PLP complex is shown in green spheres, with the substrate C4 from which a proton is abstracted is colored red. The AdoCbl cofactor is shown in blue and red for open and closed conformations, respectively. In the model for the closed formation, the adenosine moiety is in direct clash with the substrate. A reorientation to a position similar to that observed in GM (Ado in yellow) leads to a plausible van der Waals contact between the Ado C5 and the substrate.